The project elucidates the fundamental molecular design of heparins and heparan sulfates (H/HS) and studies the structure-function relations of their specific modulations of diverse normal and diseased protein and cell membrane systems, using various biological, biochemical, and physical approaches. We examine H/HS as a modulator of viral infectivity, and isolate and study Components (S-oligoS) of a heparin-mimetic, sulfated xylan drug which, as does heparin, inhibits the infectivity of HIV-1 in vitro: 1) We had revealed differential functional potencies and physical properties of the S-oligoS Components, which led to our identification and isolation of a minimal-sized, potent anti-HIV-1 S-oligoS, CpF (and purified CpF-PkII) [HD 01315-01-03]. Moreover, CpF is separated from antithrombin activity. This heparin-mimetic activity is associated with different structurally specific Components. Upscaled procedures (including automated medium pressure LC) are explored for use in obtaining a CpF-PkII drug against AIDS in humans. These procedures continue to be labor intensive, with ~8% yield. Multiple preparations to obtain sufficient purified CpF for a Phase I trial remain a primary focus and are continuing. A large scale preparation of CpF might be feasible in cooperation with the drug manufacturer with whom we have negotiated a confidential exchange. 2) Structure-function relations are studied by FTIR and quantitative proton NMR spectroscopy at 600 MHz. CpF-PKI and II are found by proton NMR to contain GlcA and Xyl in a ratio of 1:2.9 and 1:3.0, which supports our proposal [HD 01315-01- 03] of a tetraS grouping in heparin-mimetic structures (a b 1,4-linked trixyloside containing an a 1,2 D-GlcA branch). FTIR of CpF-PkII reveal sugars in the alternate as well as normal chair forms (axial as well as expected equatorial sulfates) [HD 01315-03]. Further purified PkII displays the same FTIR spectral pattern. From the proton NMR spectra we find Xyl in alternate to normal chair ratios of 1:9 in PkI and PkII. Alternate chair structures may be of significance in functional specificity since we now find that that a high mass, anti-thrombin Component (4B) displayed a 1.7-fold greater proportion of such alternate chair conformations (1:5.2), while having the same GlcA to Xyl ratio of 1:2.9. 3) The molecular mechanism underlying the inhibition of HIV-1 by S-oligoS and H/HS are investigated: Fluorescent photoactive- crosslinking CpF-PkI-probes are synthesized by direct nucleophilic addition to the aldehyde using aminonaptholsulfonic acid hydrazide (ANS) which we synthesized, or by formation of a glycamine of the S-oligoS to provide an amine for reaction with commercial photocrosslinking reagents. The ANS reaction gave good recovery of S-oligoS and ~25-50% derivatization, which will be improved by adjusting pH and T. These unique probes will assist in studies to identify and isolate a putative cell membrane S-oligoS receptor involved in viral infectivity.